Anaerobic fungi in the tortoise alimentary tract illuminate early stages of host-fungal symbiosis and Neocallimastigomycota evolution

Anaerobic gut fungi (AGF, Neocallimastigomycota) reside in the alimentary tract of herbivores. While their presence in mammals is well documented, evidence for their occurrence in non-mammalian hosts is currently sparse. Culture-independent surveys of AGF in tortoises identified a unique community, with three novel deep-branching genera representing >90% of sequences in most samples. Representatives of all genera were successfully isolated under strict anaerobic conditions. Transcriptomics-enabled phylogenomic and molecular dating analyses indicated an ancient, deep-branching position in the AGF tree for these genera, with an evolutionary divergence time estimate of 104-112 million years ago (Mya). Such estimates push the establishment of animal-Neocallimastigomycota symbiosis from the late to the early Cretaceous. Further, tortoise-associated isolates (T-AGF) exhibited limited capacity for plant polysaccharides metabolism and lacked genes encoding several carbohydrate-active enzyme (CAZyme) families. Finally, we demonstrate that the observed curtailed degradation capacities and reduced CAZyme repertoire is driven by the paucity of horizontal gene transfer (HGT) in T-AGF genomes, compared to their mammalian counterparts. This reduced capacity was reflected in an altered cellulosomal production capacity in T-AGF. Our findings provide insights into the phylogenetic diversity, ecological distribution, evolutionary history, evolution of fungal-host nutritional symbiosis, and dynamics of genes acquisition in Neocallimastigomycota.

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For amplicon datasets: Read assembly, quality control to remove any sequence with average quality score <2 5, sequences with ambiguous Data analysis bases, sequences not containing the correct barcode, sequences with more than 2bp difference in the primer sequence, and/or sequences with homopolymer stretches longer than 8bp was performed in Mathur (published software).
Transcriptomics data:RNA-seq reads were quality trimmed and de nova assembled using Trinity (v2.14.0) and default parameters (published software).Proteomics data: RAW files from the mass spectrometer were searched against the corresponding transcriptome predicted peptides database using the MaxQuant application (v2.0.2.0 57).Searches utilized MaxQuant defaults, supplemented with two additional peptide modifications: deamidation of N/Q residues, and Q cyclization to pyroglutamate.The MaxQuant "match between runs" algorithm was not used.Sequences for reversed-sequence decoy proteins and common contaminants proteins were utilized for the database searches but were removed from the final MaxQuant protein results.
Code for phylogenomic analysis is available at https://github.com/stajichlab/PHYling_unified.Code used to create other figures is available at https://github.com/nohayoussef/AGF_Tortoises For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers.We strongly encourage code deposition in a community repository (e.g.GitHub).See the Nature Portfolio guidelines for submitting code & software for further information.

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Report on the source of all seed stocks or other plant material used.If applicable, state the seed stock centre and catalogue number.If plant specimens were collected from the field, describe the collection location, date and sampling procedures.
Describe the methods by which all novel plant genotypes were produced.This includes those generated by transgenic approaches, gene editing, chemicoVradiation-based mutagenesis and hybridization.For transgenic lines, describe the transformation method, the number of independent lines analyzed and the generation upon which experiments were performed.For gene-edited lines, describe the editor used, the endogenous sequence targeted for editing, the targeting guide RNA sequence (if applicable) and how the editor wus U P.P lied.Desmbe-any-t'lt:tl'hentieation-procedtJresj-or-eadr:seed--stoek-t:Jsed--or-novef-genotype-generotedc-Beseribe-any-experiments tJsed--toassess the effect of a mutation and, where applicable, how potential secondary effects (e.g.second site T-DNA insertions, mosiacism, off-target gene editing) were examined.
fungi in the tortoise alimentary tract A total of 11 fecal samples from 11 individual available tortoises were obtained Since many of these tortoises are unaccessible in their natural habitats and many are endangered, sampling was conducted on animals housed in captivity (Oklahoma City Zoo and Hawk Hill farms, Oklahoma, USA).Only 11 total subjects were sampled due to availability Freshly deposited samples were placed in 15-or 50-ml sterile conical centrifuge tubes and transferred on ice to the laboratory, where they were stored at -202c.All samples originated from individual animals and were not adulterated during sampling with dust, dirt, or feces from other subjects.All sampled tortoises were visibly healthy during the time of sampling.Zoo personnell collected fecal samples.ITiming and spatial sc a le Only one sample was collected per animal.10 samples were obtained from OKC Zoo, and one was obtained from a local farm in Walters OK.Sampling occurred between November 2020 and March 2022Data exclusionsNo data was excluded from the analysis Reproducibility Randomization Blinding Reproducibility was assessed by comparing our results to previous studies.